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Extracellular vesicles regulate the human osteoclastogenesis: divergent roles in discrete inflammatory arthropathies

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Abstract

Objective

Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis.

Methods

Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14+ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7 days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted.

Results

EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n = 11) and RA-derived (n = 12) EXOs profoundly inhibited osteoclast differentiation (70%, p < 0.01; 65%, p < 0.01, respectively). In contrast, PsA-derived (n = 10) EXOs had a stimulatory effect (75%, p < 0.05). In cross-treatment experiments where EXOs and CD14+ cells were interchanged between the three groups, only healthy (n = 5) and RA (n = 5)-derived EXOs inhibited (p < 0.01, respectively) the generation of osteoclasts in all groups, whereas PsA (n = 7)-derived EXOs were unable to mediate this effect.

Conclusions

Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.

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Abbreviations

ACD-A:

Acid citrate dextrose-A

ACR:

American College of Rheumatology

ANOVA:

Analysis of variance

ATCC:

American Type Culture Collection

CALCR:

Calcitonin receptor

CD14:

Cluster of differentiation 14

C-FOS:

Cellular oncogene Fos

C-FMS:

Macrophage colony-stimulating factor receptor

CTSK:

Catepsin K

DAPI:

4′,6-Diamidino-2-phenylindole

DAS:

Disease activity score

DC STAMP:

Transmembrane 7 superfamily member 4

EULAR:

European League Against Rheumatism

EV:

Extracellular vesicle

EXO:

Exosome

FACS:

Fluorescence-activated cell sorting

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

IgG:

Immunoglobulin G

ISEV:

International Society for Extracellular Vesicles

LPS:

Lipopolysaccharide

M-CSF:

Macrophage colony-stimulating factor

miRNA:

Micro-ribonucleicacid

MV:

Microvesicle

NFATc1:

Nuclear factor of activated T cells cytoplasmic 1

OC:

Osteoclast

OSCAR:

Osteoclast-associated immunglobulin-like receptor

PBMC:

Peripheral blood mononuclear cell

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

RANK:

Receptor activator of nuclear factor kappa B

RANKL:

RANK ligand

RPM:

Revolution per minute

RPMI:

Roswell Park Memorial Institute medium

rSPA:

Recombinant Staphylococcal Protein A

RT:

Room temperature

SEC:

Size-exclusion chromatography

SIC:

Soluble immune complex

SLAP:

Src-like adaptor protein

TEM:

Transmission electron microscopy

TRAP:

Tartrate-resistant acid phosphatase

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Acknowledgements

This work was supported by the Hungarian Scientific Research Fund OTKA-NN111023, OTKA-NKFIH #11958; MEDINPROT and BMBS COST Action BM1202 ME HAD. Funding was provided by National Heart Program (Grant Nos. OTKA 120237, NKFIA, and KP-16-1-2016-0017).

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Authors and Affiliations

  1. Department of Genetics, Cell- and Immunobiology, Semmelweis University, Nagyvárad tér 4, 1089, Budapest, Hungary

    Nikolett Marton, Orsolya Tünde Kovács, Eszter Baricza, Edit I. Buzás & György Nagy

  2. Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

    Ágnes Kittel

  3. Department of Physiology, Semmelweis University, Budapest, Hungary

    Dávid Győri & Attila Mócsai

  4. MTA-SE “Lendület” Inflammation Physiology Research Group of the Hungarian Academy of Sciences and the Semmelweis University, Budapest, Hungary

    Dávid Győri & Attila Mócsai

  5. Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK

    Florian M. P. Meier, Carl S. Goodyear & Iain B. McInnes

  6. Department of Rheumatology, 3rd Department of Internal Medicine, Semmelweis University, Budapest, Hungary

    György Nagy

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  1. Nikolett Marton
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Contributions

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Nagy had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Correspondence to György Nagy.

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Electronic supplementary material

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18_2017_2535_MOESM1_ESM.tif

Supplementary Figure 1 Flow cytometry gate determination for ‘MV’ analysis. A, The experiment was carried out using MegaMix Beads (BioCytex, Marseille, France) and was optimized with 1 μm Silica Beads Fluo-Green (Kisker, Steinfurt, Germany). Logarithmic FSC, SSC scales were used during the measurements to visualize the ‘MVs’. ‘MVs’ are EVs of 100–1000 nm diameter. B, C, Decreased number of Annexin V positive events were detected in the ‘MV gate’ after detergent treatment. (TIFF 3711 kb)

18_2017_2535_MOESM2_ESM.tif

Supplementary Figure 2 The effect of different concentrations of SICs on the OC differentiation. SICs were generated freshly and used in different concentrations: 0.25X; 0.5X; 1X and 2X SIC treatment means that 0.25; 0.5; 1 and 2 µL of SIC samples were added to 100 µL media. The graph represents the fold increase in the number TRAP-positive cells with ≥ 3 nuclei. The values mark the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIFF 2480 kb)

18_2017_2535_MOESM3_ESM.tif

Supplementary Figure 3 The effect of size exclusion chromatography purified EXO samples on human in vitro osteoclastogenesis. Platelet free plasma samples of healthy donors (n = 2) were filtrated with 0.8 µm nanopore membranes and purified with qEV Size Exclusion Columns (Izon Science, Christchurch, New Zealand) according to manufacturer’s instructions. Then the samples were centrifuged with 100 000g, resuspended in 1*PBS and used to treat differentiating OC samples as in the previously described experiments. The graph represents the fold increase in the number of OCs (TRAP-positive cells with ≥ 3 nuclei). The values represent the mean ± SEM. *p < 0.05 ‘EXO’ refers to small EVs of approximately 100 nm diameter. (TIFF 735 kb)

18_2017_2535_MOESM4_ESM.jpg

Supplementary Figure 4 OPG, RANK, RANKL expression of EVs. EXO samples, conjugated to aldehyde/sulfate latex beads were studied by flow cytometry. Differential detergent lysis was used to study the MVs. The values represent the mean ± SEM. *p < 0.05. (JPEG 189 kb)

18_2017_2535_MOESM5_ESM.tif

Supplementary Figure 5 The detection of various CD markers on EV samples. EXO samples, conjugated to aldehyde/sulfate latex beads were studied by flow cytometry. Differential detergent lysis was used to identify the MVs. The values represent the mean ± SEM. *p < 0.05. (TIFF 13634 kb)

Supplementary material 6 (DOCX 15 kb)

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Marton, N., Kovács, O.T., Baricza, E. et al. Extracellular vesicles regulate the human osteoclastogenesis: divergent roles in discrete inflammatory arthropathies. Cell. Mol. Life Sci. 74, 3599–3611 (2017). https://doi.org/10.1007/s00018-017-2535-8

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